Modulation of the cAMP levels with a conserved actinobacteria phosphodiesterase enzyme reduces antimicrobial tolerance in mycobacteria

Abstract

Antimicrobial tolerance (AMT) is the gateway to the development of antimicrobial resistance (AMR) and is therefore a major issue that needs to be addressed.The second messenger cyclic-AMP (cAMP), which is conserved across all taxa, is involved in propagating signals from environmental stimuli and converting these into a response. In bacteria, such as M. tuberculosis, P. aeruginosa, V. cholerae and B. pertussis, cAMP has been implicated in virulence, metabolic regulation and gene expression. However, cAMP signalling in mycobacteria is particularly complex due to the redundancy of adenylate cyclases, which are enzymes that catalyse the formation of cAMP from ATP, and the poor activity of the only known phosphodiesterase (PDE) enzyme, which degrades cAMP into 5’- AMP.Based on these two features, the modulation of this system with the aim of investigating cAMP signalling and its involvement in AMT in mycobacteria id difficult.To address this pressing need, we identified a new cAMP-degrading phosphodiesterase enzyme (Rv1339) and used it to significantly decrease the intrabacterial levels of cAMP in mycobacteria. This analysis revealed that this enzyme increased the antimicrobial susceptibility of M. smegmatis mc2155. Using a combination of metabolomics, RNA-sequencing, antimicrobial susceptibility assays and bioenergetics analysis, we were able to characterize the molecular mechanism underlying this increased susceptibility.This work represents an important milestone showing that the targeting of cAMP signalling is a promising new avenue for antimicrobial development and expands our understanding of cAMP signalling in mycobacteria.

Parallel in vivo experimental evolution reveals that increased stress resistance was key for the emergence of persistent tuberculosis bacilli

“…The gene rv1339 is highly conserved in TB bacilli (Supplementary Fig. 13) and other mycobacteria and a recent study revealed that the Rv1339 protein degrades cAMP, an important signalling molecule controlling peptidoglycan metabolism 13 . Inactivation of rv1339 or expression of the mutated rv1339 allele from KC9A1 increased stress resistance in M. canettii STB-D and STB-K and reduced envelope permeability in M. smegmatis.…”

“…An identical nucleotide substitution C>T at nucleotide position 1582889, corresponding to the gene BN42_21248, was found in all clones recovered after cycle 6 (Supplementary Table 1). This mutation changed a single amino acid P96L in the protein it encodes, which is the orthologue of Rv1339, a MTB H37Rv protein, which was recently shown to encode a phosphodiesterase degrading cyclic-AMP (cAMP) 13 (Fig. 2a).…”

Triazolopyrimidines Target Aerobic Respiration in Mycobacterium tuberculosis

“…We have previously linked Rv1339 mutations to resistance to other QcrB inhibitor series, namely, the imidazopyridines and the phenoxyalkylimidazoles ( 5 , 9 ). Recent work in the related organism Mycobacterium smegmatis suggests that Rv1339 is an atypical class II cAMP phosphodiesterase that has been linked to antibiotic tolerance ( 13 ). In addition, a P94L mutation in Rv1399 led to increased persistence in animal models and increased resistance to external stress in Mycobacterium canetti , which was proposed to be due to changes in cell wall permeability ( 14 ).…”

Triazolopyrimidines target aerobic respiration in Mycobacterium tuberculosis

“…We have previously linked Rv1339 mutations to resistance to other QcrB inhibitor series, namely the imidazopyridines and the phenoxyalkylimidazoles (5,9). Recent work in the related organism Mycobacterium smegmatis suggests that Rv1339 is an atypical class II cAMP phosphodiesterase that has been linked to antibiotic tolerance (13). In addition a P94L mutation in Rv1399 led to increased persistence in animal models and increased resistance to external stress in Mycobacterium canetti, proposed to be due to changes in cell wall permeability (14).…”